【作者】 郝晓芳；

【导师】 刘在新；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2011， 博士

【摘要】 猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS)是以怀孕母猪繁殖障碍及各年龄阶段猪呼吸道疾病为主要特征的猪传染病。2006年以来,我国爆发了高致病性PRRS(highly pathogenic PRRS,HP-PRRS),使我国养猪业蒙受巨大经济损失。其病原高致病性PRRS病毒(highly pathogenic PRRSV,HP-PRRSV)在nsp2蛋白不连续缺失30个氨基酸,在我国猪场广泛流行,成为引起母猪繁殖障碍和仔猪死亡的主要病原之一。针对2007-2010年甘肃省猪场发生的母猪繁殖障碍性疾病,我们设计了PRRSV nsp2和GP5基因特异性引物,检测PRRS在甘肃省发病情况,结果大多数临床样品呈PRRSV阳性。为了进一步研究我国PRRSV的遗传变异情况,本研究选取其中7株分离株进行了PRRSV全基因组序列测定,并将其与国内外分离株进行比较分析。系统进化分析结果表明,国内PRRSV毒株可分成4个亚群,经典毒株主要位于第Ⅰ、Ⅱ和Ⅲ亚群,而高致病性毒株位于第Ⅳ亚群,该亚群与传统疫苗株RespPRRS MLV和CH-1R的亲缘关系较远,这些疫苗在预防高致病性PRRSV方面可能起不到理想的效果。第IV亚群中的高致病性PRRSV的nsp2蛋白均呈现不同程度的缺失,共有4种缺失类型的毒株,其中以不连续缺失30个氨基酸的高致病性毒株为主,这些毒株构成了从2006年至今在我国流行的优势毒株。本研究分离的07N、128、TS、XB和XIN株属于第IV亚群,具备高致病性PRRSV的分子特征;XJ株属于第Ⅲ亚群,为经典毒株;QD株在全基因组序列和nsp2蛋白序列分群中位于第Ⅰ亚群,在GP5、M和N蛋白序列分群中位于第IV亚群,推测QD株可能是经典毒株和高致病性毒株的重组毒株。各亚群均有各自独有的氨基酸序列特征,高致病性PRRSV GP5和M蛋白中的毒力相关位点保持了强毒株的序列特征,并且其中和表位发生变异,糖基化位点增多并向其下游的中和表位靠近,可能遮蔽中和表位,延迟产生中和抗体,从而逃避宿主免疫反应。以上结果表明,在甘肃省猪繁殖障碍性疾病发病猪场中PRRSV感染很普遍,并且高致病性毒株仍是危害甘肃乃至全国的主要PRRSV毒株,高致病性毒株高度同源,位于同一亚群,具有逃避宿主免疫的潜质,与之亲缘关系较远的原有传统疫苗难以防治,因此加强对PRRSV的分子流行病学调查和研制新型有效疫苗势在必行。为了调查甘肃省猪繁殖障碍性疾病中其他病毒性病原的存在情况,本研究设计了几对特异性引物,分别用于检测临床样品中的猪细小病毒(PPV)、猪圆环病毒Ⅱ型(PCV2)、猪伪狂犬病毒(PRV)以及猪瘟病毒(CSFV)等。通过对样品进行PCR或RT-PCR,结果有2份样品呈PPV阳性,有5份样品呈PCV2阳性。为了进一步研究PPV及PCV2的遗传变异情况,本研究根据GenBank中收录的PPV和PCV2的全基因组序列,分别设计了扩增PPV全基因组的引物以及扩增PCV2全基因组的引物,对阳性样品进行序列测定和分析。PPV的系统进化分析结果表明我国猪细小病毒主要分成4个主要的亚群,本研究分离的LZ株属于Ⅱ群,是最近几年来在中国常见的PPV分离株;而本研究分离的JY株属于Ⅲ群,该群是由德国PPV分离株构成的,这是我国首次分离到该群的PPV毒株。通过对PCV2的遗传进化分析,我们发现PCV2主要分成5个亚群,本研究分离的124株和XIN株属于2b亚群,XJ株和XB株属于2d亚群,而TS株属于2e亚群。以上分析结果表明,甘肃省猪繁殖障碍性疾病的病原比较复杂,并且同一种病原也存在不同的亚群,表明我们应该加强对猪繁殖障碍性疾病中各种病原的调查和分析。疫苗预防是阻止传染病在畜群蔓延的重要手段。随着HP-PRRS疫情的爆发,亟需一种能够有效预防该病的疫苗,然而传统的灭活疫苗往往不能给猪群提供足够的免疫保护力。本研究构建了共表达GP5/M蛋白的DNA疫苗以及对GP5蛋白进行修饰的ΔGP5/M DNA疫苗。小鼠试验结果表明,两种疫苗都能诱导小鼠产生较高水平的ELISA抗体,也能诱导小鼠产生病毒特异性的中和抗体,并且GP5蛋白修饰组的中和抗体水平要高于未修饰组;通过细胞免疫指标的检测,如淋巴细胞增殖试验、IFN-γ检测和小鼠T淋巴细胞亚类检测,结果显示免疫组的细胞免疫水平要高于对照组。以上结果表明本研究构建的两种表达GP5/M蛋白的DNA疫苗具有良好的免疫效果,这为研制高致病性PRRSV疫苗提供了新的思路。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome (PRRS) is characterized by reproductive failure in pregnant sows and respiratory disease in all pigs. Since 2006, a highly pathogenic PRRS (HP-PRRS) has been found in China, which caused an immense economic loss in China. The pathogen was highly pathogenic PRRSV (HP-PRRSV), which was found a dis-continuous deletion of 30 aa in nsp2 protein. The HP-PRRSVs are still prevalent in swine herd in China and become a main cause of reproductive failure of sows and death in young pigs.In order to investigate the pathogens causing the reproductive failure of sows in Gansu province in 2007-2010, the specific primer pairs against PRRSV nsp2 and ORF5 gene were designed, according to the conserved sequences of PRRSV genome. The PCR detection showed that most of the clinical samples were PRRSV-positive. In order to well understand the genetic variation of PRRSV in China, we selected 7 strains sequenced in this study and some other Chinese reference strains to analyze the phylogenetic relationship and amino acid aligment. The phylogenetic analysis shows that Chinese PRRSV strains could be divided into 4 sub-groups. The classical strains mainly located in sub-group I,ⅡandⅢ, while the HP-PRRSVs located in sub-group IV. The HP-PRRSV was in far relation to traditional vaccine strains RespPRRS MLV and CH-1R, which indicated that these vaccines might be of malfunction in prevention of HP-PRRSV infection. The nsp2 protein of sub-group IV had some different types of deletion, among which most of the HP-PRRSVs were characterized by a dis-continuous deletion of 30 aa in nsp2 protein. Viruses of this type were the dominant strains in China. Our PRRSV strains 07N, 128, TS, XB and XIN belonged to sub-group IV and were of HP-PRRSV; strain XJ belonged to sub-groupⅢ, which was classical strain; Interestingly, QD strain belonged to sub-groupⅠin whole genome tree and nsp2 protein tree, while belonged to sub-group IV in GP5, M, and N protein trees. It may be concluded that QD strain was a recombinant strain between classical strain and HP-PRRSV strain. From the amino acid alignment, all sub-groups were characterized by different amino acids. The HP-PRRSV had the same characterization with virulent strains in residues responsible for virulence in GP5 and M protein. Moreover, amino acid mutation appeared in neutralizing epitope. The glycosylation sites began to accumulate and were close to the downstream neutralizing epitope. Hence, the glycosylation sites could cover up the neutralizing epitope, detain the production of neutralizing antibody, and enable the virus to escape from the immune response of host. In all, the HP-PRRSV infection in Gansu province is very common and it needs further reseaches for molecular epidemiology of PRRSV and development of new vaccine against PRRSV in China.In order to investigate the other viral pathogens related to swine reproductive failure in Gansu province, several primer pairs were designed to detect the viral pathogens, e.g. porcine parvovirus (PPV), porcine circovirus typeⅡ(PCV2), pseudorabies virus (PRV), and classical swine fever virus (CSFV). The detection results showed that two samples were PPV-positive, while five samples were PCV2-positive. No PRV- or CSFV-positive sample was detected in all samples. To further understand the genetic characterization of PPV and PCV2 isolated in Gansu province, the genomes of PPV and PCV2 were amplified with specific primers, respectively. The phylogenetic analysis of PPV showed that the PPV strains could be divided into 4 sub-groups. LZ strains isolated in this study belonged to II sub-group, which was common PPV strains in China; while JY strains belongs to III sub-group, which was composed of German PPV strains. This was the first isolation of Germany-like PPV in China. Phylogenetic analysis of PCV2 showed that the PCV2 strains could be divided into 5 sub-groups. The strains 124 and XIN isolated in this study belonged to sub-group 2b; strains XJ and XB belonged to sub-group 2d; while strain TS belonged to sub-group 2e. It can be concluded that the viral pathogens related to swine reproductive failure in Gansu Province are complicate. Molecular epidemiology should be conducted in order to well understand these epizootics in Gansu province.Vaccine prevention is a key measure to prevent the epizootics in domestic animals. With the outbreak of HP-PRRS in China, it is badly in need of an effective vaccine to prevent the prevalence of HP-PRRSV in China. However, the traditional inactivated vaccines usually did not provide enough immunologic potency to swine herds. In this study, DNA vaccine co-expressing GP5/M or modifiedΔGP5/M was constructed using pcDNA3.1 vector. The experiment in mouse model showed that both kinds of vaccines could induce high level of ELISA antibodies in mouse, as well as the virus-specific neutralizing antibodies. Moreover, the level of neutralizing antibody of modified GP5 group was higher than that of ordinary GP5 group. Cellular immune detections, e.g., lymphocyte proliferation assay, IFN-γdetection, and T-lymphocyte subsets detection, showed that both kinds of vaccines had good immune effect, compared with the control. The DNA vaccines co-expressing GP5 and M of HP-PRRSV would provide a new approach to develop the HP-PRRSV vaccine.更多还原